Return to study ST002474 main page
MB Sample ID: SA247791
Local Sample ID: | 36-week db/db+aav9-RDH10-2 |
Subject ID: | SU002564 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Genotype Strain: | db/m; db/db; db/db+retinol; db/db+retinoic acid; db/db+AAV9-RDH10; RDH10-cKO; RDH10fl/fl |
Age Or Age Range: | 32-week; 36-week; 21-week |
Gender: | Male |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002564 |
Subject Type: | Mammal |
Subject Species: | Mus musculus |
Taxonomy ID: | 10090 |
Genotype Strain: | db/m; db/db; db/db+retinol; db/db+retinoic acid; db/db+AAV9-RDH10; RDH10-cKO; RDH10fl/fl |
Age Or Age Range: | 32-week; 36-week; 21-week |
Gender: | Male |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
36-week db/db+aav9-RDH10-2 | SA247791 | FL031161 | db/db | Genotype |
36-week db/db+aav9-RDH10-2 | SA247791 | FL031161 | aav9-rdh11 | Treatment |
Collection:
Collection ID: | CO002557 |
Collection Summary: | Mice were fasted for 12 hours before sampling and testing. All animal experiments were approved by the Animal Care and Ethics Committee of Zhongshan School of Medicine, Sun Yat-sen University, and followed the National Institutes of Health Guidelines on the Care and Use of Animals (the protocol number is SYSU-IACUC-2019-B027) |
Sample Type: | Cardiac tissue |
Treatment:
Treatment ID: | TR002576 |
Treatment Summary: | RDH10-cKO mice: RDH10-cKO mice, which contain both RDH10fl/fl and MYH6-iCre, were bred by RDH10fl/fl mice and MYH6-iCre mice and were injected tamoxifen intraperitoneally (50 mg/kg, T2859, Sigma -Aldrich, St. Louis, MO) for 5 consecutive days from 5 weeks of age. Age-matched male RDH10fl/fl mice that also received TMX injections served as normal controls for RDH10-cKO mice. Animal treatments: Mice in the Rol treatment group received Rol gavage (800 IU/each, 17772, Sigma-Aldrich, St. Louis, MO) every two days from 8 weeks of age. Mice in the atRA treatment group received atRA intraperitoneal injection (5 mg/kg body weight, R2625, Sigma-Aldrich, St. Louis, MO) daily from 8 weeks of age. Gene therapy: A recombinant AAV9 vector carrying the mouse RDH10 sequence (AAV9-RDH10, DZ-AAV-Rdh10-OE, Dongze, Hanbio Inc, Shanghai, China) was used to overexpress RDH10. 0.8*10^11 vg/per animal of AA9-RDH10 was transferred into T2DM mice, respectively, by tail vein injection at the age of 16 weeks. |
Sample Preparation:
Sampleprep ID: | SP002570 |
Sampleprep Summary: | Sample preparation Homogenized the heart tissue in 200μL of cold NaCl solution (0.9%) for 5 seconds, add 100μL retinol-D4 (IR-23012, IsoSciences, Ambler, PA, USA) as internal standard for Rol and atRA, and retinyl acetate (46958, Sigma-Aldrich, St. Louis, MO) as internal standard for retinyl esters, then homogenize for another 5 seconds. For Rol and at RA, 1mL formic acid n-hexane solution (1%) was added to the homogenizing to facilitate phase separation by a 5-minutes vortex and 5-min centrifugation (16,200 g). The supernatants were collected in a new tube and blown dry with nitrogen followed by re-dilution with 100μL 70% methanol. For retinyl esters, 24 μL 0.5 M NaOH solution was added to tissue homogenate, followed by 1 mL n-hexane. The mixture was votexed 5 min and centrifuged at 16,200 g for 5 min. The supernatants were collected in a new tube and blown dry with nitrogen followed by re-dilution with 100μL 100% methanol. The processes above should be protected from the light. |
Combined analysis:
Analysis ID | AN004041 |
---|---|
Analysis type | MS |
Chromatography type | Reversed phase |
Chromatography system | Sciex Triple Quad TM 4500MD MS |
Column | Phenomenex Kinetex C18 (50 x 2.1mm,1.7um) |
MS Type | ESI |
MS instrument type | QTRAP |
MS instrument name | ABI Sciex 5500 QTrap |
Ion Mode | POSITIVE |
Units | ng/ml |
Chromatography:
Chromatography ID: | CH002989 |
Instrument Name: | Sciex Triple Quad TM 4500MD MS |
Column Name: | Phenomenex Kinetex C18 (50 x 2.1mm,1.7um) |
Column Temperature: | 25 |
Flow Gradient: | initial conditions 75% B; from 0 to 3.5min linear increase to 95% B; between 3.5 and 4.5min 95% B was retained; at 4.6 min back to initial conditions with 75% B; finally, 75% B was held from 4.6 to 5.9min |
Flow Rate: | 0.2ml/min |
Solvent A: | 40% acetonitrile/30% methanol/30% water; 0.1 % formic acid |
Solvent B: | 55% acetonitrile/30% methanol/15% water; 0.1 % formic acid |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS003788 |
Analysis ID: | AN004041 |
Instrument Name: | ABI Sciex 5500 QTrap |
Instrument Type: | QTRAP |
MS Type: | ESI |
MS Comments: | The MS conditions were as follows: electrospray ionization (ESI) under positive mode; nebulizer gas: nitrogen; curtain gas,30 psi; ion spray voltage, 4000V; temperature, 400ºC; gas 1 and gas 2, 35 and 40 psi, respectively; collision gas, 10 psi. The parameters of the mass spectrometer were optimized, and the multiple reaction monitoring (MRM) transitions of retinol, all-trans-retinoic acid, as well as D4-retinol were chosen as 269.2>93.1, 273.1>94.0, and 301.2>123.1, respectively. The MRM transitions for retinyl esters were chosen as 329.3>269.3 for retinyl acetate, 524.4>268.1 for retinyl palmitate (16:0), 552.5>268.2 for retinyl stearate (18:0), 522.4>268, retinyl palmitoleate (16:1), and retinyl oleate (18:1). The quantification was performed using the calibration curve. |
Ion Mode: | POSITIVE |